Antiapoptotic functions of Bcl-2, Bcl-xL, and potentially other family members, including Mcl-1 and Bcl-w, confer marked resistance to the cytotoxic effects of available anticancer agents. In addition, Bcl-2 survival proteins are overexpressed in comparison to normal tissue counterparts in a significant subset of common cancers. Low-basal Bcl-xL expression is a strong negative predictor of cell survival with diverse classes of chemotherapeutic agents in the sixty cell lines included in the National Cancer Institute (NCI) anticancer drug screen. For these reasons, small-molecule inhibitors of Bcl-2 and Bcl-xL are being considered as a goal for molecularly targeted cancer therapy. A major challenge in developing therapeutic inhibitors for Bcl-2-related survival proteins is the expression of these targets in many normal cell types (Hockenbery et al. Proc Natl Acad Sci USA 1991; 88:6961-65; Park J R, et al. Blood 1995; 86:868-76). The dependence of healthy tissues on the Bcl-2 family of antiapoptotic proteins for cell viability may narrow the therapeutic window for these agents. 2-methoxy antimycin A (2-MeAA) represents the first of a novel class of inhibitors that display gain-of-function cytotoxicity, defined as enhanced killing in a cell line overexpressing a Bcl-2 survival protein target compared with an isogenic control cell line. (Manion et al. Current Opinion in Investigational Drugs 2006; 7:1077-84; and Schwartz et al. Mol Cancer Ther 2007; 6:2073-80).
Based on the gain-of-function model, cancer cells with high endogenous levels of Bcl-xL would be predicted to exhibit greater sensitivity to 2-MeAA than cancers with low Bcl-xL expression. Bcl-xL expression shows a positive correlation with sensitivity to 2-MeAA when comparing five cancer cell lines with the highest Bcl-xL mRNA expression in the NCI anticancer drug screen to five cell lines with lowest Bcl-xL expression, i.e., 2-MeAA is most cytotoxic against cells with the highest Bcl-xL expression. In contrast Bcl-xL expression levels show a negative correlation with standard therapeutic agents, i.e., standard therapeutics are less cytotoxic against cells with high Bcl-xL expression levels. Schwartz 2007. Mesothelioma cell lines with high expression of Bcl-xL and Bcl-2 show in vitro and in vivo sensitivity to 2-MeAA. Thus, this class of activity (i.e., gain-of-function inhibitors) may expand the potential of Bcl-2 inhibitors beyond chemosensitization while also providing an improved therapeutic index.
Overexpression of Bcl-xL in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule Bcl-xL inhibitors. Based on knockout studies, non-neoplastic cells also require Bcl-xL survival functions, particularly when challenged with cytotoxic agents. One Bcl-xL inhibitor, 2-MeAA, was found to be cytotoxic in cells with excess exogenous Bcl-xL but had less cytotoxicity in isogenic cell line pairs having basal levels of Bcl-xL expression (Tzung et al. Nature New Biol 2001; 3:183-91; Hockenbery et al. U.S. Pat. No. 7,241,804, issued Jul. 10, 2007; and Hockenbery et al. US 2005/0239873, filed Jan. 14, 2005). This selectivity, characteristic of a gain-of-function mechanism, is not shared by other known Bcl-xL inhibitors, including BH3I-2, HA14-1, ABT-737, gossypol, or the stapled BH3 helical peptide SAHB-BID (Schwartz 2007). Also in contrast to other Bcl-xL inhibitors, gain-of-function Bcl-xL inhibitors can be combined with a standard inducer of apoptosis, staurosporine, to enhance selective cytotoxicity toward Bcl-xL-overexpressing cells. (Id.)
Small-molecule, gain-of function Bcl-xL inhibitors, were identified showing characteristic preferential cytotoxicity against cells overexpressing Bcl-xL cells. These include NSC 310343 inhibitor with gain-of-function activity, (Id.) and others. (Wu et al. WO 08/021,250, filed Aug. 10, 2007, and Schwartz et al. WO 08/021,211, filed Aug. 10, 2007). Other compounds have been shown to be inhibitors of proliferative disorders, including cancer (Hirth et al. U.S. Pat. No. 5,700,823; Beachy et al. WO 05/033048; Okada et al. U.S. Pat. No. 5,807,880). Methods for detecting cell apoptosis and methods for screening potential therapeutic compounds which inhibit or stimulate apoptosis have been described (Siman et al. U.S. Pat. No. 6,048,703; and Soto et al. U.S. Pat. No. 6,939,679; Tomei et al. WO 99/03054).
It would be desirable to identify other compounds that may be effective in inducing apoptosis in cells where apoptosis is inappropriately regulated while causing minimal cytotoxicity in normal, otherwise isogenic cells.